Protein kinase C (PKC) is an important regulatory enzyme whose physiological activation requires it to become membrane-bound. This occurs via the simultaneous presence of calcium, an acidic phospholipid such as phosphatidyl serine (PS), and a diglyceride. The presence of the latter is of great regulatory significance because it is the product of polyphosphatidylinositol turnover. The diglyceride requirement can be satisfied by structurally diverse tumor promoters such as the phorbol esters, teleocidin, and the debromoaplysiatoxins. It is likely that PKC is an important target for the tumor promoters. In addition, PKC plays an important role in platelet aggregation, cardiac function, and neural function. The major focus of this grant is to understand the mechanism of the novel activation process of PKC. Of particular interest is the unravelling the structural basis for PKC activator function. On the one hand, PKC is exceedingly selective with respect to the chemical structure of diglyceride activators. At the same time, structurally diverse tumor promoters belonging to the diterpene, peptide, and macrolide series , can all potently activate the enzyme. These observations are reconciled here with the presentation of a new unifying structural hypothesis on PKC activators which are based on experiments described in the progress report. This new hypothesis makes predictions concerning novel diglyceride and peptide analogs which should potently interact with the kinase. These molecules will be synthesized and studied as putative PKC activators and inhibitors. Finally, novel cyclic PS analogs will be prepared to study the specificity of the phospholipid binding-site of PKC. Complimentary studies on the nature of activator binding-site are also anticipated. The PKC regulatory domain binding-site will be covalently labeled with activator-based photoaffinity probes and the labeled region will be sequenced. Taken together, these studies should bring us closer to an understanding of the regulation of PKC at a molecular level and the general issue of how proteins can interact with and bind to membranes. Finally, the structural information obtained here will serve to map the PKC diglyceride binding-site and can be used as a starting point to prepare potent inhibitors of the enzyme.